Methods to screen for transcription factor-coactivator interactions

Abstract

This invention provides methods for modulating gene expression at the transcriptional level. In particular, the methods involve tethering a transcriptional coactivator to a DNA binding domain that is specific for a target nucleic acid sequence and contacting the coactivator with a transcription factor. The transcription factor triggers or represses transcription mediated by the coactivator. Methods for identifying compounds that are able to modulate gene expression are also provided.

What is claimed is: 
     
       1. A method of identifying test compounds that modulate transcription of a gene of interest by modulating the triggering function of a transcription factor, the method comprising: i) contacting a nucleic acid with a tethered coactivator, said nucleic acid comprising a reporter gene operably linked to a response element, and said tethered coactivator comprising: a) a polypeptide that comprises an activation function from a transcriptional coactivator; and   b) a DNA binding moiety that specifically binds to the response element;     ii) contacting the tethered coactivator with a transcription factor polypeptide that comprises an activation function of a transcription factor that is involved in modulating expression of the gene of interest in its natural milieu, wherein the transcription factor polypeptide is contacted with a test compound prior to contacting the tethered coactivator; and   iii) detecting expression of the reporter gene wherein an alteration of the expression of the reporter gene as compared to expression of the reporter gene where the transcription factor polypeptide is not contacted with the test compound indicates that said test compound modulates the triggering function of the transcription factor.   
     
     
       2. The method according to claim 1, wherein the method is carried out in a cell. 
     
     
       3. The method according to claim 2, wherein the transcription factor polypeptide is provided by expression of a heterologous gene encoding the transcription factor polypeptide in a cell. 
     
     
       4. The method according to claim 1, wherein said modulating results in activation of said reporter gene. 
     
     
       5. The method according to claim 1, wherein the response element is heterologous to the gene of interest. 
     
     
       6. The method according to claim 1, wherein the transcription factor is a nuclear hormone receptor. 
     
     
       7. The method according to claim 1, wherein the test compound interferes with the ability of the nuclear hormone receptor to become associated with its natural hormone. 
     
     
       8. The method according to claim 1, wherein the test compound acts as a mimetic of a natural hormone in its ability to modulate activity of the nuclear hormone receptor. 
     
     
       9. The method according to claim 6, wherein the transcription factor is an estrogen receptor. 
     
     
       10. A method of identifying test compounds that modulate an indirect estrogen response by modulating the triggering function of an estrogen receptor, the method comprising: i) contacting a nucleic acid with a tethered coactivator, said nucleic acid comprising a reporter gene operably linked to an AP-1 site, and said tethered coactivator comprising: a) a polypeptide that comprises an activation function from a transcriptional coactivator, and   b) a DNA binding moiety that specifically binds to the AP-1 site;     ii) contacting the tethered coactivator with the estrogen receptor, wherein the estrogen receptor is contacted with a test compound prior to contacting the tethered coactivator; and   iii) detecting expression of the reporter gene wherein an alteration of the expression of the reporter gene as compared to expression of the reporter gene where the estrogen receptor is not contacted with the test compound indicates that said test compound modulates an indirect estrogen response by modulating the triggering function of the estrogen receptor.   
     
     
       11. The method according to claim 10, wherein the estrogen receptor is provided by expression of a heterologous gene encoding said estrogen receptor. 
     
     
       12. The method according to claim 10, wherein said modulating results in activation of said reporter gene. 
     
     
       13. The method according to claim 10, wherein the test compound interferes with the ability of the receptor to become associated with estrogen. 
     
     
       14. The method according to claim 10, wherein the test compound acts as a mimetic of a natural hormone in its ability to modulate activity of the estrogen receptor.